Protein Kinase C (PKC) is activated at the cell membrane in response to several products of lipid metabolism. Different PKC isozymes respond to different cellular environments and modulators to transduce distinct signals involved in cellular regulation; however, the differences and mechanisms are poorly understood. To develop more specific modulators for therapeutic use and cellular studies, it is necessary to better understand the structure and activation of these isozymes at a membrane surface. Our general hypotheses (not mutually exclusive) are that PKC modulators may bind to specific sites on PKC isozymes and/or they may modulate lipid domain composition and thereby alter PKC membrane binding and activation. To understand how these modulators work, our goals are to obtain information on the structure of PKC at a membrane surface, the structure of the lipid domains with which it interacts, and the effects of various PKC modulators on both. In Aim I we will use 2D crystallization of PKC on lipid monolayers to elucidate the structure of PKC isozymes in the presence and absence of various inhibitors, activators and substrates. Already we have obtained 3 - 5 crystal forms each of PKC alpha and delta on monolayers composed of phospholipid: diacylglycerol mixtures. Conditions for generating the crystals will be optimized, the relationship between the several lattice forms of each isozyme will be evaluated, and 3D reconstructions of the 2D data will be made. We will attempt to model the membrane-bound form based on homologous domains from other proteins and on the modelled structure of an inactive PKC. Effects of several PKC activators, inhibitors and substrates on crystal formation and conformation of PKC will be examined and sites for those that bind PKC will be investigated. In Aim II, we will examine the effects of some of the same lipid modulators (lysolipids, ceramides) on lipid domain formation and on PKC activation and membrane binding using differential scanning calorimetry, atomic force microscopy, and 2D crystal formation. Collectively this information should facilitate design of new PKC modulators that directly interact with sites on PKC and/or that alter lipid properties required for PKC activation.